(+)-trans-4-(1-aminoethyl)-1-(4-pyridycarbamoyl)-cyclohexane and method for promoting neural growth in the central nervous system and in a patient at a site of neuronal lesion

ABSTRACT

The invention relates to an antagonist of one or more of Rho family members having ability to elicit neurite outgrowth from cultured neurons in an assay method which includes culturing neurons on a substrate that incorporates a growth-inhibiting amount of Rho family member and exposing the cultured neurons to a candidate Rho family member antagonist agent to permit neuron growth. Candidates which elicit neurite outgrowth from the cultured neurons are thus identified as Rho family antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 09/184,572 filed on Nov. 2, 1998.

FIELD OF INVENTION

This invention relates to the regulation of growth of neurons in theCentral Nervous System.

BACKGROUND

Following trauma in the adult central nervous system (CNS) of mammals,injured neurons do not regenerate their transected axons. An importantbarrier to regeneration is the axon growth inhibitory activity that ispresent in CNS myelin and that is also associated with the plasmamembrane of oligodendrocytes, the cells that synthesize myelin in theCNS (see Schwab M. E., et al., (1993) Ann. Rev. Neurosci. 16, 565-595,for review). The growth inhibitory properties of CNS myelin have beendemonstrated in a number of different laboratories by a wide variety oftechniques, including plating neurons on myelin substrates or cryostatsections of white matter, and observations of axon contact with matureoligodendrocytes (Schwab, M. E., et al., (1993) Annu. Rev. Neurosci. 16,565-595). Therefore, it is well documented that adult neurons cannotextend neurites over CNS myelin in vitro.

It has also been well documented that removing myelin in vivo improvesthe success of regenerative growth over the native terrain of the CNS.Regeneration occurs after irradiation of newborn rats, a procedure thatkills oligodendrocytes and prevents the appearance of myelin proteins(Savio and Schwab, (1990) Neurobiology 87, 4130-4133). After such aprocedure in rats is combined with a corticospinal tract lesion, somecorticospinal axons regrow long distances beyond the lesions. Also, in achick model of spinal cord repair, the onset of myelination correlateswith a loss of its regenerative ability of cut axons (Keirstead, et al.,(1992) Proc. Nat. Acad. Sci. (USA) 89, 11664-11668). The removal ofmyelin with anti-galactocerebroside and complement in the embryonicchick spinal cord extends the permissive period for axonal regeneration.These experiments demonstrate a good correlation between myelination andthe failure of axons to regenerate in the CNS.

Myelin inhibits axon growth because it contains at least severaldifferent growth inhibitory proteins. It has been well documented by usand by others that myelin-associated glycoprotein (MAG) has potentgrowth inhibitory activity, both in vitro and in vivo (McKerracher, L.,et al., (1994) Neuron 13, 805-811; Mukhopadhyay, G., et al., (1994)Neuron 13, 805-811; Li, M., et al., (1996) J Neurosci. Res. 46, 404-414;Schafer, M., et al., (1996) Neuron 16, 1107-1113). A high molecularweight inhibitory activity has been characterized by Schwab andcollaborators, and neutralization of this activity with the IN-1antibody allows some axons to regenerate in white matter (Schwab, M. E.,et al., (1993) Ann. Rev. Neurosci. 16 565-595; Bregman, B., et al.,(1995) Nature 378, 498-501.). We also have evidence that there is anadditional growth inhibitory protein in myelin (Xiao, Z., et al., (1997)Soc. Neurosci. Absts. 23, 1994). Clearly, there are multiple inhibitoryproteins that stop axon regeneration in mammalian CNS myelin.

In addition to the myelin-derived inhibitors there are also other growthinhibitory molecules expressed in the adult mammalian CNS. Tenacin is agrowth inhibitory protein that is expressed in some unmyelinated regionsof the CNS (Bartsch, U., et al., (1994) J Neurosci. 14, 4756-4768) andafter lesion tenascin is expressed by astrocytes that border the lesionsite (Ajemain and David (1994) J Comp. Neurol. 340, 233-242). Alsogrowth inhibitory proteins that are proteoglycans are expressed byreactive astrocytes, and these proteins form a barrier to regenerationat the glial scar (McKeon and Silver (1995) Exp. Neurol. 136, 32-43).

While axons damaged in the CNS in vivo do not typically regrow, therehave been some reports of long distance axon extension in adult whitematter. Such growth has been observed following transplantation ofgrafted neural tissue (Wictorin, K., et al., (1990) Nature 347, 556-558;Davies, S. J. A., et al., (1994) J Neurosci. 14, 1596-1612; Isacson, O.and Deacon, T. W. (1996) Neuroscience 75, 827-837), suggesting thatembryonic neurons primed for rapid extension of axons may be lesssusceptible to growth inhibition. Some embryonic neurons are notsusceptible to MAG (Mukhopadhyay, G., et al., (1994) Neuron 13,805-811), but most embryonic neurons are inhibited by the other myelininhibitors (Schwab, M. E., et al., (1993) Ann. Rev. Neurosci. 16,565-595). Therefore, in the cases when axons are able to extend onmyelin, signaling through intracellular pathways may play an importantrole in stimulating, or blocking the inhibition of axon growth. Forexample, it is known that laminin is able to stimulate rapid neuritegrowth (Kuhn, T. B., et al., (1995) Neuron 14, 275-285), and we havedocumented that when laminin is present in sufficient concentration,neurites can extend directly on myelin substrates. These findingssuggest the possibility that the stimulation of the integrins, thereceptors for laminin, is sufficient to allow axon growth on myelin.Similarly, it has been documented that when the adhesion molecule L1 isexpressed ectopically on astrocytes, it can partially overcome theirnon-permissive substrate properties (Mohajeri, M. H., et al., (1996)Eur. J. Neurosci. 8, 1085-1097). Therefore, neurons can, underappropriate conditions, grow axons on inhibitory substrates, suggestingthat the balance of positive to negativegrowth cues is a criticaldeterminant for the success or failure of axon regrowth after injury.

Growth inhibitory proteins typically cause growth cone collapse, aprocess that causes dramatic rearrangements to the growth conecytoskeleton (Bandtlow, C. E., et al., (1993) Science 259, 80-83; Fan,J., et al., (1993) J Cell Biol. 121, 867-878; Li, M., et al., (1996) JNeurosci. Res. 46, 404-414). One family of proteins that has beenimplicated in receptor-medicated signaling to the cytoskeleton is thesmall GTPases of the Rho family (Hall, A. (1996) Ann. Rev. Cell Biol.10, 31-54). In non-neuronal cells it has been clearly documented thatmutations in Rho family members that include Rho, Rac and cdc42, affectadhesion, actin polymerization, and the formation of lamellipodia andfilopodia, which are all processes important to motility (Nobes, C. D.and Hall, A. R. (1995) Cell 81, 53-62). There is now good evidence thatmembers of the Rho family regulate axon outgrowth in development.Mutations in Rho-related family members block the extension of axons inDrosophila (Luo, L., et al., (1994) Genes Dev. 8, 1787-1802) and disruptaxonal pathfinding in C. elegans (Zipkin, I. L., et al., (1997) Cell 90,883-894). More recently it has been shown that the guidance moleculecollapsin acts through a Rac-dependent mechanism (Jin, Z. andStrittmatter, S. M. (1997) J. Neurosci. 17, 6256-6263). In transgenicmice that express constitutively active Rac in Purkinje cells, there arealterations in the development of axon terminals and dendriticarborizations (Luo, L., et al., (1996) Nature 379, 837-840). Consistentwith the observations in vivo, it was found that dominant negative Racexpressed in PC12 cells disrupts neurite outgrowth in response to NGF(Hutchens, J. A., et al., (1997) Molec. Biol. Cell 8, 481-500). Also,treatment of PC12 cells with lysophosphatidic acid, a mitogenicphospholipid, causes neurite retraction that is mediated by Rho (Tigyi,G., et al., (1996) J. Neurochem. 66, 537-548). Therefore, differentmembers of the Rho family can exert distinct effects on neurite growth,and in PC12 cells the activation of Rho is correlated with growth conecollapse. In non-neuronal cells, Rho participates in integrin-dependentsignaling (Laudanna, C., et al., (1996) Science 271, 981-983; Udagawa,T. and McIntyre, B. W. (1996) J. Biol. Chem. 271, 12542-12548). Thepossibility that Rho might play a role within the myelin-derived growthinhibitory system has been studied (Jin, Z. and Strittmatter, S. M.(1997) J. Neurosci. 17, 6256-6263). It was concluded, however, that theinhibitory effects of myelin are not mediated by Rho family members.

A need remains for a means of inactivating the multiple inhibitoryproteins present in myelin that prevent axonal regrowth after injury inthe CNS.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY OF THE INVENTION

The present invention relates to antagonists and inhibitors to membersof the Rho family of proteins and diagnostic, therapeutic, and researchuses for each of these aspects. In particular, members of the Rho familyof proteins serve as a therapeutic target to foster regrowth of injuredor degenerating axons in the CNS.

In accordance with the present invention, a preferred embodiment relatesto antagonists and inhibitors of members of the Rho family of proteinsand their use as a means of blocking a common signaling pathway used bythe diverse growth inhibitory molecules. The antagonists and inhibitorsmay be mutated forms of Rho and biologically active (Rhofamily-inhibitory) fragments, peptides, C3 and biologically active (Rhofamily-inhibitory) fragments, or small molecules such as Y-27632.

In yet a further aspect of the present invention, Rho family memberproteins can be used to design small molecules that antagonize andinhibit Rho family proteins, to block inhibition of neurite outgrowth.In another aspect of the present invention Rho family members can beused to design antagonist agents that suppress the myelin growthinhibitory system. These antagonist agents can be used to promote axonregrowth and recovery from trauma or neurodegenerative disease.

In a further aspect of the present invention, inhibitors of the Rhofamily of proteins can be used to block inhibition of neurite outgrowthand to suppress the myelin growth inhibitory system. Such inhibitorscould block exchange of the GTP/GDP cycle of Rhoactivation/inactivation.

A further embodiment involves a method of suppressing the inhibition ofneuron growth, comprising the steps of delivering to the nerve growthenvironment, antibodies directed against Rho family members in an amounteffective to reverse said inhibition.

In accordance with another aspect of the present invention, there isprovided an assay method useful to identify Rho family member antagonistagents that suppress inhibition of neuron growth, comprising the stepsof:

-   a) culturing neurons on a growth permissive substrate that    incorporates a growth-inhibiting amount of a Rho family member; and-   b) exposing the cultured neurons of step a) to a candidate Rho    family member antagonist agent in an amount and for a period    sufficient prospectively to permit growth of the neurons;    thereby identifying as Rho family antagonists the candidates of    step b) which elicit neurite outgrowth from the cultured neurons of    step a).

In accordance with another aspect of present invention, there isprovided a method to suppress the inhibition of neuron, comprising thesteps of delivering, to the nerve growth environment, a Rho familyantagonist in an amount effective to reverse said inhibition.

In another embodiment, kinases activated by Rho, such as Rho-associatedkinase, are antagonist candidates. Thus, compounds such as Y-27632 (U.S.patent Ser. No. 04/997,834), that block Rho-associated kinase activity,thereby inactivating the Rho signaling pathway, are also embodiments ofthis invention. Thus, the use of other compounds within this family ofcompounds as described in U.S. patent Ser. No. 04/997,834 that inhibitRho kinase are also considered within the scope of this invention.

In another embodiment, the nucleic acids encoding Rho family members canbe used in antisense techniques and therapies.

In yet another embodiment, a kit is provided comprising componentsnecessary to conduct the assay method useful to screen Rho familyantagonist agents.

Various other objects and advantages of the present invention willbecome apparent from the detailed description of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of treatment with C3 to stimulate neurite outgrowthon inhibitory MAG substrates. A) PC12 cells plated on MAG remainedrounded and did not extend neurites. B) Cells plated on MAG in thepresence of C3 grew neurites. C) PC12 cells plated on polylysine (PLL)substrates as a positive control.

FIG. 2 shows the role of integrins in overriding growth inhibition bymyelin. The anti-α1 integrin function blocking antibody, 3A3, was usedto determine if integrin function is necessary for laminin to overridegrowth inhibition by myelin or MAG. For experiments on myelin substrates(A-D), cells were fluorescently labeled with DiI, and plated on myelin(A), polylysine (B), or myelin +1 μg laminin (C and D). Control IgG wasadded to samples A-C, the 3A3 antibody to D. Neurites do not extend onmyelin but grow on laminin or mixed laminin/myelin substrates. When 3A3is added, laminin no longer overrides growth inhibition by myelin.Panels (E-H) show by phase contrast cells plated on recombinant MAG (E),laminin (F), or recombinant MAG plus laminin (G and H), with controlantibody (E-G) or with 3A3 (H). Integrin function is needed to overridegrowth inhibition by MAG.

FIG. 3 presents the results of studies in which PC12 cells transfectedwith dominant negative Rho extend short neurites on MAG substrates.Mock-transfected PC12 cells (a,c,e) or cells transfected withdominant-negative Rho (b,d,f) were plated on laminin (a,b) or MAG (c-f).MAG inhibits neurite outgrowth (c), but dominant negative Rho cellsspread on MAG and some cells extend short neurites (d). Treatment withC3 further stimulates neurite outgrowth on MAG from both lines of cells(e,f).

FIG. 4 shows activation of Rho on MAG substrates. Activated Rho isassociated with the plasma membrane. To determine if activated Rho wasdetected under conditions where PC12 cells do not grow neurites, cellswere grown in suspension or plated on MAG or collagen substrates. Twohours later the plasma membranes were purified, the proteins separatedby SDS PAGE, and the proteins transferred to nitrocellulose and stainedwith Ponceau S. Rho A was detected on the blots by immunoreactivity withanti-RhoA antibody. Immunoreactivity was strongest when cells were grownin suspension or when cells were plated on MAG. Therefore, Rho A is moreactive when cells are kept in suspension or plated on MAG than whenplated on growth-permissive collagen.

FIG. 5 shows treatment of retinal neurons with C3 stimulates neuritegrowth on polylysine and MAG substrates. On nMAG substrates neuritegrowth is inhibited (a), but after C3 treatment retinal neurons platednMAG substrates extend neurites (b). Growth of neurites from retinalneurons plated on PLL (c) Bar, 50 μm.

FIG. 6 demonstrates ADP-ribosylation of Rho by C3 detected in culturedcells. PC12 cells or retinal neurons were cultured in the presence (+)or absence of C3 (−) for two days. The cells were lysed, and 10 μg ofprotein from each sample was separated on a 11% acrylamide gel. Theproteins were transferred to nitrocellulose, probed with mouse anti-RhoAantibody and anti-mouse-HRP antibody, and revealed by a chemiluminescentreaction (top panel). The membranes were then reprobed with rabbitanti-Cdc42 and anti-rabbit alkaline phosphatase and revealed withNTB/BCIP color reaction. Treatment of cells with C3 results in anADP-ribosylation-induced decrease in the mobility of RhoA. The mobilityof Cdc42 does not change with C3 treatment.

FIG. 7 illustrates methods used to study the effect of C3 on injuredoptic nerve. FIG. 7 a shows the optic nerve was removed from the sheathprior to crushing with 10.0 sutures (top) and C3 was applied in Gelfoamand Elvax tubes (red bars) immediately following optic nerve crush(middle). The retinal ganglion cell axons were detected by anterogradelabeling with cholera toxin and immunodetection of the cholera toxin inlongitudinal sections of the optic nerve (bottom). FIGS. 7 c, 7 d, 7 e,and 7 f show treatment of crushed optic nerve with C3 stimulatesregenerative growth of retinal ganglion cells axons. (c) Longitudinal 15μm section of a buffer-treated control optic nerve showing the failureof RGC axons to cross the injured region; (d,e) Longitudinal 15 μmsections of two different optic nerves treated with C3 showinganterogradely-labeled axons extending past the crush (arrows). The siteof crush is indicated with arrowheads; (f) Higher magnification view of(e) showing the twisted growth of regenerating axons. Bar, 100 μm(c,d,e) and 50 μm in f. FIG. 7 b shows quantitation of axon regenerationacross the site of lesion. Representation of regeneration observed indifferent animals. For each animal, the maximum number of axons observedin a single 14 μm section was counted at different distances from thesite of the crush. Each point represents one animal, but animals withgrowth past 500 μm are also represented at the shorter distances. Largenumbers of regenerating fibers (>10/section) were observed to cross thelesion after C3 treatment compared to treatment with PBS.

FIG. 8 shows the effect of Y27632 on neurite outgrowth of primaryneurons plated on inhibitory substrates compared with C3 . P0 retinalganglion cells (RGCs) were examined after 48 hours on the following testsubstrates: Polylysine (PLL) chondroitin sulfate proteoglycan (CSPG) ormyelin in the presence or absence of C3 or Y27632. Shown is the averageRGC neurite length after treatment with 50 μg/ml C3 or Y27632 (densehatching), 25 μg/ml C3 or Y27632 (light hatching), or no treatment(white).

FIG. 9 shows the measurement of regeneration distances in mice withspinal cord injury alone, in mice treated with collagen and fibrin ascontrols, in mice animals treated with C3 in collagen or fibrin gels,and in mice treated with Y27632 in fibrin. Each point represents oneanimal. The circles are animals examined at 3 weeks to one month, thetriangles animals examined 3 months after spinal cord injury.

FIG. 10 shows an analysis of functional recovery. Modified BBB scores ofC3-treated (black circles), Y27632-treated (black triangles),fibrin-treated (hatched circles), and untreated (open circles) mice toevaluate recovery of locomotion for one month following dorsalover-hemisection. Each point represents the average of 10-11 animals±SEMfor controls and C3 experiments, or 5 animals for Y27632.

DETAILED DESCRIPTION OF THE INVENTION

This invention arises from the discovery that Rho family members are keymolecules in regulating inhibition by myelin proteins, and by MAG. Thus,this invention provides the advantage of identifying an intracellulartarget, Rho family members, for all of the multiple inhibitory proteinsthat must be inactivated to allow for growth on myelin. The method ofthis invention provides for inactivation of Rho family members, therebystimulating neurite growth on growth inhibitory substrates. Therefore,antagonists that inactivate Rho family members in vivo should allow axonregeneration in the injured or diseased CNS.

This invention provides for the use of Rho, or proteins related to Rhoas therapeutic targets for agents designed to block growth inhibition bymyelin or myelin proteins. One embodiment pertains to the use of Rhoantagonists that foster axon regeneration in the central nervous system.The therapeutic agent or antagonist can be small molecules, proteins orpeptides, or any agent that binds to Rho or its family members toinactivate this pathway. Another embodiment pertains to the use of theRho regulatory pathway as a target for Rho antagonists. This pathwayinvolves the GDP/GTP exchange proteins (GEPs). Rho has twointerconvertible forms, GDP-bound inactive, and GTP-bound active forms.The GEPs promote the exchange of nucleotides and thereby constitutetargets for regulating the activity of Rho. In another embodiment GDPdissociation inhibitors (GDIs) inhibit the dissociation of GDP from Rho,and thereby prevent the binding of GTP necessary for the activation ofRho. Therefore, GDIs are targets for agents that regulate Rho activity.The GTP-bound active Rho can be converted to the GDP-found inactive formby a GTPase reaction that is facilitated by its specific GTPaseactivating protein (GAP). Thus, another embodiment pertains to the useof GAPs as targets for the regulation of Rho activity. Anotherembodiment pertains to the fact that Rho is found in the cytoplasmcomplexed with a GTPase inhibiting protein (GDI). To become active, Rhobinds GTP and is translocated to the membrane. Thus, agents that affectRho binding to the plasma membrane are also considered within the scopeof this invention. Yet another embodiment pertains to the observationthat a bacterial mon-ADP ribosyltransferase, C3 transferase, ribosylatesRho to inactivate the protein. Thus this embodiment pertains to the useof C3 transferase to inactivate Rho and stimulate axon growth. Likewise,other bacterial toxins, such as toxins A and B, with relatedRho-inhibitory activity are considered to be within the scope of thisinvention. Moreover, various mutations of the Rho protein can createdominant negative Rho, which can interfere with the biological activityof endogenous Rho in neurons. Thus, yet a further embodiment of thisinvention pertains to the use of dominant negative forms of Rho, used toinactivate Rho, to foster axon growth.

“Antagonist” refers to a pharmaceutical agent which in accordance withthe present invention which inhibits at least on biological activitynormally associated with Rho family members, that is blocking orsuppressing the inhibition of neuron growth. Antagonists which may beused in accordance with the present invention include withoutlimitation, one or more Rho family members fragment, a derivative of Rhofamily members or of a Rho family members fragment, an analog of Rhofamily members or of a Rho family members fragment or of saidderivative, and a pharmaceutical agent, and is further characterized bythe property of suppressing Rho family members-mediated inhibition ofneurite outgrowth. Preferred antagonists include: mutated forms of Rho,such as Rho wherein the effector Domain, A-37, has been mutated toprevent GTP exchange; the ADP-ribosyl transferase C3 and biologicallyeffective fragments that antagonize Rho family members in one of theassays of this invention; and compounds such as Y-27632 that antagoniseRho-associated-kinase (Somiyo, 1997, Nature, 389:908-910; Uehata, etal., 1997, Nature 389:990-994; U.S. Pat. No. 4,997,834).

The antagonist of Rho family members in accordance with the presentinvention is not limited to Rho family members or its derivatives, butalso includes the therapeutic application of all agents, referred hereinas pharmaceutical agents, which alter the biological activity of the Rhofamily members protein such that inhibition of neurons or their axon issuppressed.

The term “effective amount” or “growth-promoting amount” refers to theamount of pharmaceutical agent required to produce a desired antagonisteffect of the Rho family members biological activity. The preciseeffective amount will vary with the nature of pharmaceutical agent usedand may be determined by one or ordinary skill in the art with onlyroutine experimentation.

As used herein, the Rho family of proteins comprises, but is not limitedto rho, rac, cdc42 and their isotypes, such as RhoA, RhoB, RhoC, as wellas Rho-associated kinases that are expressed in neural tissue. Othermembers of the Rho family that are determined and whose inhibition ofactivity allows for neurite outgrowth are contemplated to be part ofthis invention. (See, for example, Katoh, H., et al., J. Biol. Chem.,273:2489-2492, 1998; van Leeuwen, F., et al., J. Cell Biol.,139:797-807, 1997; Matsui et al., EMBO J. 15:2208-2216, 1996; Amano etal., Science, 275:1308; Ishizaki, T. et al., (1997) FEBS Lett.,404:118-124).

As used herein, the terms “Rho family member biological activity” refersto cellular events triggered by, being of either biochemical orbiophysical nature. The following list is provided, without limitation,which discloses some of the known activities associated withcontact-mediated growth inhibition of neurite outgrowth, adhesion toneuronal cells, and promotion of neurite out growth from new born dorsalroot ganglion neurons.

As used herein, the term “biologically active”, or reference to thebiological activity of Rho family members, or polypeptide fragmentthereof, refers to a polypeptide that is able to produce one of thefunctional characteristics exhibited by Rho family members or itsreceptors described herein. In one embodiment, biologically activeproteins are those that demonstrate inhibitory growth activities centralnervous system neurons. Such activity may be assayed by any method knownto those of skill in the art.

The term C3 refers to C3 ADP-ribosyltransferase, a specific Rhoinactivator. A preferred representative example is C3ADP-ribosyltransferase, a 23 KDa exoenzyme secreted from certain strainsof types C and D from Clostridium botulinum, which specificallyADP-ribosylates the rho family of these GTP-binding proteins. ThisADP-ribosylation occurs at a specific asparagine residue in theirputative effector domain, and presumably interferes with theirinteraction with a putative effector molecule downstream in signaltransduction. Numerous references describing these compounds can befound in Methods in Enzymology, Vol 256, Part B, Eds.; W. E. Balch, C.H. Der, and A. Hall; Academic Press, 1995, for e.g. Pgs. 196-206, 207 etseq, 184-189 and 174 et seq.

Based on the present evidence that Rho family members can affect growthinhibitory protein signals in myelin, the means exist to identify agentsand therapies that suppress myelin-mediated inhibition of nerve growth.Further, one can exploit the growth inhibiting properties of Rho familymembers, or Rho family members antagonists, to suppress undesired nervegrowth. Without the critical finding that a Rho family member has growthinhibitory properties, these strategies would not be developed.

Rho Family Member Antagonists and Assay Methods to Identify Rho FamilyMembers Antagonists

In one embodiment, Rho family member antagonists will be inhibitors ofGTPase activity. The GTP/GDP cycle of Rho family membersactivation/inactivation is regulated by a number of exchange factors.Compounds that block exchange, thereby inactivating Rho family membersare preferred embodiments of this invention.

In another embodiment suitable Rho family member antagonist candidatesare developed comprising fragments, analogs and derivatives of Rhofamily members. Sequences for Rho family members are known, such asthose described: Chardin, P., et al., (1988) Nucleic Acids Research,16:2717; Yeramian, et al., (1987) Nucleic Acids Research, 15: 1869).Such candidates may interfere with Rho family members-mediated growthinhibition as competitive but non-functional mimics of endogenous Rhofamily members. From the amino acid sequence of Rho family members andfrom the cloned DNA coding for it, it will be appreciated that Rhofamily members fragments can be produced either by peptide synthesis orby recombinant DNA expression of either a truncated domain of Rho familymembers, or of intact Rho family members could be prepared usingstandard recombinant procedures, that can then be digested enzymicallyin either a random or a site-selective manner. Analogs of Rho familymembers or Rho family members fragments can be generated also byrecombinant DNA techniques or by peptide synthesis, and will incorporateone or more, e.g. 1-5, L- or D-amino acid substitutions. Derivatives ofRho family members, Rho family members fragments and Rho family membersanalogs can be generated by chemical reaction of the parent substance toincorporate the desired derivatizing group, such as N-terminal,C-terminal and intra-residue modifying groups that have the effect ofmasking or stabilizing the substance or target amino acids within it.

In specific embodiments of the invention, candidate Rho family memberantagonists include those that are derived from a determination of thefunctionally active region(s) of Rho family member. The antibodiesmentioned above and any others to be prepared against epitopes in Rhofamily members, when found to be function-blocking in in vitro assays,can be used to map the active regions of the polypeptide as has beenreported for other proteins (for example, see Fahrig, et al., (1993)Europ. J Neurosci., 5, 1118-1126; Tropak, et al., (1994) J Neurochem.,62, 854-862). Thus, it can be determined which regions of Rho familymembers GTPases recognized by substrate molecules that are involved ininhibition of neurite outgrowth. When those are known, syntheticpeptides can be prepared to be assayed as candidate antagonists of theRho family members effect. Derivatives of these can be prepared,including those with selected amino acid substitutions to providedesirable properties to enhance their effectiveness as antagonists ofthe Rho family members candidate functional regions of Rho familymembers can also be determined by the preparation of altered forms ofthe Rho family members domains using recombinant DNA technologies toproduce deletion or insertion mutants that can be expressed in variouscell types as chimeric proteins. All of the above forms of Rho familymembers, and forms that may be generated by technologies not limited tothe above, can be tested for the presence of functional regions thatinhibit or suppress neurite outgrowth, and can be used to design andprepare peptides to serve as antagonists.

In accordance with an aspect of the invention, the Rho family memberantagonist is formulated as a pharmaceutical composition which containsthe Rho family member antagonist in an amount effective to suppress Rhofamily member-mediated inhibition of nerve growth, in combination with asuitable pharmaceutical carrier. Such compositions are useful, inaccordance with another aspect of the invention, to suppress Rho familymember-inhibited nerve growth in patients diagnosed with a variety ofneurological disorders, conditions and ailments of the PNS and the CNSwhere treatment to increase neurite extension, growth, or regenerationis desired, e.g., in patients with nervous system damage. Patientssuffering from traumatic disorders (including but not limited to spinalcord injuries, spinal cord injuries, spinal cord lesions, surgical nervelesions or other CNS pathway lesions) damage secondary to infarction,infection, exposure to toxic agents, malignancy, paraneoplasticsyndromes, or patients with various types of degenerative disorders ofthe central nervous system can be treated with such Rho family membersantagonists. Examples of such disorders include but are not limited toStrokes, Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacobdisease, kuru, Gerstman-Straussler syndrome, scrapie, transmissible minkencephalopathy, Huntington's disease, Riley-Day familial dysautonomia,multiple system atrophy, amylotrophic lateral sclerosis or Lou Gehrig'sdisease, progressive supranuclear palsy, Parkinson's disease and thelike. The Rho family members antagonists may be used to promote theregeneration of CNS pathways, fiber systems and tracts. Administrationof antibodies directed to an epitope of Rho family member, or thebinding portion thereof, or cells secreting such antibodies can also beused to inhibit Rho family member function in patients. In a particularembodiment of the invention, the Rho family members antagonist is usedto promote the regeneration of nerve fibers over long distancesfollowing spinal cord damage.

In another embodiment, the invention provides an assay method adapted toidentify a Rho family member antagonists, that is agents that block orsuppress the growth-inhibiting action of Rho family members. In its mostconvenient form, the assay is a tissue culture assay that measuresneurite out-growth as a convenient end-point, and accordingly uses nervecells that extend neurites when grown on a permissive substrate. Nervecells suitable in this regard include neuroblastoma cells of the NG108lineage, such as NG108-15, as well as other neuronal cell lines such asPC12 cells (American Type Culture Collection, 12301 Parklawn Drive,Rockville, MD 20852 USA, ATCC Accession No. CRL 1721), humanneuroblastoma cells, and primary cultures of CNS or PNS neurons takenfrom embryonic, postnatal or adult animals. The nerve cells, forinstance about 10³ cells-microwell or equivalent, are cultured on agrowth permissive substrate, such as polylysine or laminin, that isover-layed with a growth-inhibiting amount of Rho family members. TheRho family members incorporated in the culture are suitablemyelin-extracted Rho family members, although forms of Rho familymembers other than endogenous forms can be used provided they exhibitthe Rho family members property of inhibiting neuron growth when addedto a substrate that is otherwise growth permissive.

In this assay, candidate Rho family member antagonists, i.e., compoundsthat block the growth-inhibiting effect of Rho family members, are addedto the Rho family member-containing tissue culture preferably in amountsufficient to neutralize the Rho family member growth-inhibitingactivity, that is between 1.5 and 15 μg of Rho family members antagonistper well containing a density of 1000 NG108-15 cells/well cultured for24 hr. in Dulbecco's minimal essential medium. After culturing for aperiod sufficient for neurite outgrowth, e.g. 3-7 days, the culture isevaluated for neurite outgrowth, and antagonists are thereby revealed asthose candidates, which elicit neurite outgrowth. Desirably, candidatesselected as Rho family members antagonists are those which elicitneurite outgrowth to a statistically significant extent, e.g., in atleast 50%, more desirably at least 60%, e.g. 70%, per 1,000 culturedneurons.

Other assay tests that could be used include without limitation thefollowing: 1) The growth cone collapse assay that is used to assessgrowth inhibitory activity of collapsin (Raper, J. A., and Kapfhammer,J. P., (1990) Neuron, 2, 21-29; Luo, L., et al., (1993) Cell 75,217-227) and of various other inhibitory molecules (Igarashi, M., etal., (1993) Science 259 77-79) whereby the test substance is added tothe culture medium and a loss of elaborate growth cone morphology isscored. 2) The use of patterned substrates to assess substratepreference (Walter, J. et al., (1987) Development 101, 909-913; Stahl,et al., (1990) Neuron 5, 735-743) or avoidance of test substrates(Ethell, D. W., et al., (1993) Dev. Brian Res. 72, 1-8). 3) Theexpression of recombinant proteins on a heterologous cell surface, andthe transfected cells are used in co-culture experiments. The ability ofthe neurons to extend neurites on the transfected cells is assessed(Mukhopadhyay et al., (1994) Neuron 13, 757,767). 4) The use of sectionsof tissue such as sections of CNS white matter, to assess molecules thatmay modulate growth inhibition (Carbonetto, S., et al., (1987) J.Neuroscience 7, 610-620; Salvo, T. and Schwab, M. E., (1989) JNeurosci., 9:1126-1133). 5) Neurite retraction assays whereby testsubstrates are applied to differentiated neural cells for their abilityto induce or inhibit the retraction of previously extended neurites(Jalnink, et al., (1994) J Cell Bio. 126, 801-810; Sudan, H. S., et al.,(1992) Neuron 8, 363-375; Smalheiser, N., (1993) J Neurochem. 61,340-342). 6) The repulsion of cell-cell interactions by cell aggregationassays (Kelm, S., et al., (1994) Current Biology 4, 965-972;Brady-Kainay, S., et al., (1993) J Cell Biol. 4, 961-972). 7) The use ofnitrocellulose to prepare substrates for growth assays to assess theability of neural cells to extend neurites on the test substrate(Laganeur, C. and Lemmon, V., (1987) PNAS 84, 7753-7757; Dou, C-L andLevine, J. M., (1994) J. Neuroscience 14, 7616-7628).

Diagnostic, Therapeutic and Research Uses for Rho Family MemberAntagonists

Rho family member antagonists have uses in diagnostics. Such moleculescan be used in assays to detect, prognose, diagnose, or monitor variousconditions, diseases, and disorders affecting neurite growth extension,invasiveness, and regeneration. Alternatively, the Rho family memberantagonists may be used to monitor therapies for diseases and conditionswhich ultimately result in nerve damage; such diseases and conditionsinclude but are not limited to CNS trauma, (e.g. spinal cord injuries),infarction, infection, malignancy, exposure to toxic agents, nutritionaldeficiency, paraneoplastic syndromes, and degenerative nerve diseases(including but no limited to Alzheimer's disease, Parkinson's disease,Huntington's Chorea, amyotrophic lateral sclerosis, progressivesupra-nuclear palsy, and other dementias). In a specific embodiment,such molecules may be used to detect an increase in neurite outgrowth asan indicator of CNS fiber regeneration. For example, in specificembodiments, altered levels of Rho family members activity in a patientsample containing CNS myelin can be diagnostic marker for the presenceof a malignancy, including but not limited to glioblastoma,neuroblastoma, and melanoma, or a condition involving nerve growth,invasiveness, or regeneration in a patient.

Useful for nerve growth suppression are pharmaceutical compositions thatcontain, in an amount effective to suppress nerve growth, Rho familymember antagonist in combination with an acceptable carrier. CandidateRho family members antagonists include fragments of Rho family membersthat incorporate the ectodomain, including the ectodomain per se andother N-and/or C-terminally truncated fragments of Rho family members orthe ectodomain, as well as analogs thereof in which amino acids, e.g.from 1 to 10 residues, are substituted, particularly conservatively, andderivatives of Rho family members or Rho family members fragments inwhich the N- and/or C-terminal residues are derivatized by chemicalstabilizing groups.

In a preferred embodiment, mutated forms of Rho family members are usedas antagonists. One key example is Rho with a mutated effector domain,A-37, which prevents GTP exchange. Various other mutations of the Rhoprotein that create dominate negative Rho which can interfere with thebiological activity of endogenous Rho in neurons are considered asantagonists within the scope of this invention to inactivate Rho,thereby fostering growth of neurons.

In another preferred embodiment GDP dissociation inhibitors (GDIs),which inhibit the dissociation of GDP from Rho, and thereby prevent thebinding of GTP necessary for the activation of Rho are used asantagonists.

In yet another preferred embodiment, GTPase activating protein (GAP)which facilitates the conversion of the GTP-bound active Rho to theGDP-bound inactive form forms the target for regulation of Rho activity.Thus, compounds that activate GAP, thereby facilitating the conversionof active Rho into inactive Rho.

In still another preferred embodiment, compounds that affect Rho bindingto the plasma membrane, thereby decreasing the activity of Rho are alsoconsidered Rho antagonists of this invention. In this case, the targetdesign is based on the knowledge that Rho is found in the cytoplasmcomplexed with GTPase inhibiting protein (GDI). To become active, Rhobinds GTP and is translocated to the membrane. Thus, agents that induceRho binding to the plasma membrane would decrease Rho activity.

In specific embodiments of the invention, candidate Rho family membersantagonists include specific regions of the Rho family members molecule,and analogs or derivatives of these. These can be identified by usingthe same technologies described above for identification of Rho familymembers regions that serve as inhibitors of neurite outgrowth.

The Rho family members related derivatives, analogs, and fragments ofthe invention can be produced by various methods known in the art. Themanipulations, which result in their production can occur at the gene orprotein level. For example, Rho family members-encoding DNA can bemodified by any of numerous strategies known in the art (Maniatis etaL., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y, 1982), such as by cleavage atappropriate sites with restriction endonuclease(s), subjected toenzymatic modifications if desired, isolated, and ligated in vitro.

Additionally, the Rho family members-encoding gene can be mutatedin-vitro or in-vivo for instance in the manner applied for production ofthe ectodomain, to create and/or destroy translation, initiation, and/ortermination sequences, or to create variations in coding regions and/orform new restriction endonuclease sites or destroy preexisting ones, tofacilitate further in-vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to, in-vitrosite directed mutagenesis (Hutchinson, et al., (1978) J Biol. Chem. 253,6551), use of TAB™ linkers (Pharmacia), etc.

For delivery of Rho family members antagonists, various known deliverysystems can be used, such as encapsulation in liposomes or semipermeablemembranes, expression in suitably transformed or transfection glialcells, oligodendroglial cells, fibroblasts, etc. according to theprocedure known to those skilled in the are (Lindvall, et al., (1994)Curr. Opinion Neurobiol. 4, 752-757). Linkage to ligands such asantibodies can be used to target delivery to myelin and to othertherapeutically relevant sites in-vivo. Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, oral, and intranasal routes, and transfusioninto ventricles or a site of operation (e.g. for spinal cord lesions) ortumor removal. Likewise, cells secreting Rho family members antagonistactivity, for example, and not by way of limitation, hybridoma cellsencapsulated in a suitable biological membrane may be implanted in apatient so as to provide a continuous source of Rho family membersinhibitor.

Therapeutic Uses of Rho Family Antagonists

In an embodiment, antagonists, derivatives, analogs, inhibitors of Rhofamily members can be used in regimens where an increase in neuriteextension, growth, or regeneration is desired, e.g., in patients withnervous system damage. Patients suffering from traumatic disorders(including but not limited to spinal cord injuries, spinal cord lesions,or other CNS pathway lesions), surgical nerve lesions, damage secondaryto infarction, infection, exposure to toxic agents, malignancy,paraneoplastic syndromes, or patients with various types of degenerativedisorders of the central nervous system can be treated with suchinhibitory protein antagonists. Examples of such disorders include butare not limited to Alzheimer's Disease, Parkinson's Disease,Huntington's Chorea, amyotrophic lateral sclerosis, progressivesupranuclear palsy and other dementias. Such antagonists may be used topromote the regeneration of CNS pathways, fiber systems and tracts.Administration of antibodies directed to an epitope of, (or the bindingportion thereof, or cells secreting such as antibodies) can also be usedto inhibit Rho family members protein function in patients. In aparticular embodiment of the invention, antibodies directed to Rhofamily members may be used to promote the regeneration of nerve fibersover long distances following spinal cord damage.

Various delivery systems are known and can be used for delivery ofantagonists or inhibitors of Rho family members and related molecules,e.g., encapsulation in liposomes or semipermeable membranes, expressionby bacteria, etc. Linkage to ligands such as antibodies can be used totarget myelin associated protein-related molecules to therapeuticallydesirable sites in vivo. Methods of introduction include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, oral, and intranasal routes, and infusion into ventriclesor a site of operation (e.g. for spinal cord lesions) or tumor removal.

In addition, any method which results in decreased synthesis of Rhofamily members may be used to diminish their biological function. Forexample, and not by way of limitation, agents toxic to the cells whichsynthesize Rho family members and/or its receptors (e.g.oligodendrocytes) may be used to decrease the concentration ofinhibitory proteins to promote regeneration of neurons.

EXAMPLES Example 1

This example demonstrates in vitro evidence that Rho family members areresponsible for regulating the neuronal response to MAG. In particular,this demonstrates that the small GTPase Rho regulates the response toMAG. PC12 cells were planted on polysine (PLL), laminin, or MAGsubstrates and exposed to NGF to stimulate neurite growth. PC12 cellsdifferentiated neurites on PLL and laminin substrates, but on MAGsubstrates the cells remained rounded and did not grow neurites.

The addition of the ADP-ribosyl transferase C3 from Clostidiumbotulinum, that efficiently inactivates Rho family members withoutaffecting Rac and cdc42 (Udagawa, T. and McIntyre, B. W. (1996) J. Biol.Chem. 271, 12542-12548), allowed the cells to extend neurites on MAGsubstrates. In addition this example demonstrates neurite growth fromPC12 cells transfected with a dominant negative N19RhoA construct. Onlaminin and PLL substrates the N19 RhoA PC12 cells grew neurites thatwere longer than the mock-transfected controls. Moreover, N19 RhoA PC12cells were able to extend neurites when plated on MAG substrates.Therefore, the inactivation of Rho stimulates neurite outgrowth andallows neurite extension on MAG substrates. These results implicate Rhoin signaling growth inhibition by MAG.

Cell Culture

We obtained PC12 cells from three different sources: from Dr. PhilBarker (Montreal Neurological Institute); from the ATCC (obtained fromW. Mushinsky, McGill), and from Gabor Tigyi, (University of Tennessee)and we found that all lines of cells were inhibited by both myelin andMAG. PC12 cells were grown in Dulbecco's modified eagle's medium (DMEM)with 10% horse serum and 5% fetal bovine serum. PC12 cells stablytransfected with constitutively active and dominant negative RhoAconstructs were kindly provided by Dr. G. Tigyi University of Tennessee,Memphis, USA). The three cell lines used included a mocktransfected cellline, and constitutively active RhoA (V14GRhoA) cell line, and adominant negative RhoA (N19TRhoA) cell line. Transfected PC12 cell lineswere maintained in the growth medium containing 400 mg/L G418. For celldifferentiation experiments, cells were plated on appropriate substratesin DMEM with 1% fetal bovine serum and 100 ng/ml nerve growth factor.For experiments on mixed substrata (laminin/MAG or laminin/myelin), PC12were plated in DMEM with 1% lipid free-BSA in the presence or theabsence of 50 mg/ml of an irrelevant antibody or of a purified functionblocking antibody (clone 3A3) against the rat a1b1 integrin (a gift ofS. Carbonetto). PC12 cell differentiation experiments were done in96-well plates in duplicate, and each experiment was repeated a minimumof three times.

To culture cerebellar granule cells, 3-4 rats from P3 to P7 weredecapitated. The cerebellum was removed and placed in MEM-HEPES whereunderlying tissue and the meninges was removed. The cerebellum was cutinto small pieces and treated with 0.125% trypsin in MEM-HEPES for 20′at 37 C. The tissue was then triturated with a fire polished pasteurpipette to break up any clumps of tissue. The cells were spun down at1500 rpm for 10′, and the pellet was resuspended in MEM-HEPES with 2 mMEDTA. The cell suspension was placed on an isoosmotic percoll gradientwith 60% and 35% percoll, centrifuged for 15′ at 2300 rpm, and theinterface between the 60% and 35% percoll was collected. Cells werewashed once, and resuspended in DMEM with 10% FBS, vitamins, andpenicillin/streptomycin in the presence or absence of 20 mg/ml C3transferase. Cells were placed in 4-chamber, chamber slides coated withpoly-l-lysine or laminin and treated with spots of MAG or myelin. 200,00cells per chamber were plated.

Preparation of Growth Substrates

Poly-l-lysine was obtained from Sigma (St. Louis, M.o). Laminin wasprepared from EHS tumors (Paulsson and Lindblom (1994) Cell biology: Alaboratory handbook, Academic Press, pp 589-594) and collagen from rattails (Greene, et al., (1987) Meth. Enzymology 147, 207-216). Myelin wasmade from bovine brain corpus callosum, and native MAG was purified frommyelin after extraction in 1% octylglucoside and separation by ionexchange chromatography (McKerracher, L., et al., (1994) Neuron 13805-811). This native MAG has some additional proteins, including sometenascin (Xiao, Z., et al., (1997) Neurosci. Abstr. 23 1994).Recombinant MAG was made in baculovirus as described (McKerracher, L.,et al., (1994) Neuron 13, 805-811).

Test substrate were prepared as uniform substrates in 96-well plates or4-chambered slides, or as spots on 18 mm glass coverslips. First,poly-L-lysine was coated by incubation of 100 mg/ml for 3 hours at 37 C,and the wells or coverslips were washed with water and dried. Lamininsubstrates were prepared by incubating 25 mg/ml laminin on poly-L-lysinecoated dishes for 3 hours at 37 C. Solid MAG or myelin substrates wereprepared by drying down MAG overnight, or incubating at 10 mg/ml myelinsolution for 3 hours on polylysine coated substrates. For 96-wellplates, 1-4 mg of either recombinant MAG (rMAG) or of native MAG perwell was used. For mixed laminin/myelin or lamin/MAG substrata, 8 mg ofinhibitory proteins and 10 mg of laminin were dried down on 96-wellplates precoated with polylysine. For 4-chambered chamber slides, 40 mgMAG per chamber used, and for 100 mm plates 0.6-1 mg of MAG was drieddown. Spots of MAG on coverslips were generated by plating of 2 mg/mlrecombinant MAG on polylysine for 3-4 hours in a humid chamber at 37° C.Collagen substrates were made by incubating 10-15 mg/ml of rat tailcollagen for 3 hours at 37° C.

Immunocytochemistry

PC12 cells were visualized by phase contrast microscopy, or followinglabeling with the lipophilic fluorescent dye, DiI (McKerracher, L., etal., (1994) Neuron 13, 805-811). Granule cells were visualized byimmunocyteochemistry. Following 12-24 hours in culture, cells were fixedfor 30′ at room temperature in 4% paraformaldehyde, 0.5% glutaraldehyde,0.1 M phosphate buffer. Following fixation, cells were washed 3×5′ withPBS and then blocked for 1 hour at room temperature in 3% BSA, 0.1%Triton-X 100. Granule cell cultures were incubated overnight with apolyclonal anti-rMAG antibody (called 57A++) to label MAG spots. The MAGantibody was detected using an FITC conjugated secondary antibody.Rhodamine conjugated phalloidin was diluted 1:200 with the secondaryantibody to label granule cell actin filaments.

C3 Transferase Preparation and Use

The plasmid pGEX2T-C3 coding for the GST-C3 fusion protein was obtainedfrom A. Hall (London). Recombinant C3 was purified as described byDillon and Feig (Met. Enzymology, (1994), 256, pp 174-184). After fusionprotein cleavage by thrombin, thrombin was removed by incubating theprotein solution 1 hour on ice with 100 ml of p-aminobenzamidineagarose-beads (Sigma). The C3 solution was desalted on PD10 column(Pharmacia) with PBS, and sterilized through a 0.22 mm filter. The C3concentration was evaluated by Lowry assay (DC protein assay, Bio-Rad)and toxin purity was controlled by SDS-PAGE analysis.

To test the effect of C3 on the outgrowth on PC12 cells, C3 transferasewas scrape loaded into the cells before plating on appropriatesubstrates. Cells were grown to confluence in serum containing media in6 well plates. Cells were washed once with scraping buffer (114 mM KC1,15 mM NaCl, 5.5 mM MgCl_(2,) 10 mM Tris-HC1). Cells were then scrapedwith a rubber policeman into 0.5 ml scraping buffer in the presence orabsence of 20 mg/ml C3 transferase. The cells were pelleted, andresuspended in 2 ml DMEM, 1% FBS, and 50 ng/ml nerve growth factorbefore plating. 10 mg/ml C3 was added to scrape loaded cells. Cells weredifferentiated for 48 hours then fixed in 4% paraformaldehyde, 0.5%glutaraldehyde, 0.1 M PO₄ buffer.

Membrane Translocation Assay for RhoA

PC12 cells were collected and resuspended in DMEM, 0.1% BSA, 50 ng/mlNGF, then plated on 100 mm dishes coated with collagen or MAG, or leftin suspension. Two hours later, cells were washed with ice coldPBS+protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/mlantipain, 1 mg/ml pepstatin). Cells were then scraped into 5 mlPBS+protease inhibitors, and the cells were pelleted and washed withPBS+protease inhibitors. The cell pellets were mechanically homogenizedby 25 strokes in a glass-teflon homogenizer, the homogenate centrifugedfor 20 min at 8,000 rpm, and the cell debris in the pellet wasdiscarded. The supernatant was centrifuged for 1 hour at 100,000×g toseparate members and cytosolic fractions. Membrane pellets were washed1× with PBS+protease inhibitors and resuspended in PBS with 0.5% SDS,and 50-100 mg of membrane protein was analyzed by SDS-PAGE on 12% gels.Gels were transferred to Protran nitrocellulose membrane and stainedwith Ponceau S. Blots were blocked for 1 hour in 5% skim milk in TBS,and probed overnight with Rho A antibody diluted 1:200 in 1.5% skim milkTBS. Rho A antibody was detected by using an alkaline phosphataseconjugated secondary antibody and an alkaline phosphatase detection kit(Gibco-BRL).

Growth Inhibition of PC12 Cells and its Modulation by NGF and Laminin

PC12 cells typically extend neurites in response to NGF, but when platedon myelin substrates the cells remain round and do not extend neurites(Moskowitz, P. F., et al., (1997) J. Neurosi. Rec. 34, 129-134.) (FIG.2). MAG is a potent inhibitor of axon growth present in myelin. Weobserved that PC12 cells plated on substrates of MAG also remainedrounded (FIG. 1), a finding in contrast to a report that PC12 cells arenot responsive to MAG (Bartsch, U., et al., (1995) Neuron 15,1375-1381). To further examine the response of PC12 cells to MAG, weplated three different lines of PC12 cells on both native andrecombinant MAG substrates in the presence of NGF. All of the lines ofPC12 cells showed reduced cell spreading, and most cells remainedrounded without neurites. However, with increasing time, some neuriteswere able to extend on MAG substrates (see below). We also observed thatdifferent preparations of MAG can differ in their potency to inhibitneurite growth, and that the activity of MAG is reduced or lost uponfreeze-thaw.

Laminin is known to override completely, growth inhibition of NG108cells by myelin (David, S., et al., (1995) J. Neurosci. Res. 42,594-602). Similarly, we found that PC12 cells are able to extendneurites on mixed myelin and laminin substrates or on mixed laminin/MAGsubstrates (FIG. 2). To determine if signaling through integrins isresponsible for overriding growth inhibition by myelin, we used theintegrin function blocking antibody 3A3 raised against the α1 subunitextracellular domain. Previous studies have documented that α1β1integrin is the dominant integrin expressed by PC12 cells, and that the3A3 antibody blocks PC12 cell neurite growth on laminin (Tomaselli, K.J., et al., (1990) Neuron 5, 651-662). We plated PC12 cells on mixedmyelin and laminin substrates, in the presence of the 3A3 antibody, orwith a non-specific IgG antibody as a control. The 3A3 antibody blockedneurite extension on both and laminin and the mixed myelin/lamininsubstrates (FIG. 2). On MAG or on myelin substrates the cells remainedrounded. The observation that the 3A3 antibody restores growthinhibition on mixed substrates demonstrates that laminin does notoverride growth inhibition by masking the inhibitory domain of MAG, butthat signals elicited through integrins receptors are responsible.

Effect of C3 Transferase on PC12 Cells

To investigate possible intracellular targets that may override growthinhibition by myelin: and by MAG, we focused on the small GTPase Rhowhich is known to play a role in convergent-signaling pathways thataffect morphology and motility (Hall, A., (1996) Ann. Rev. Cell Biol. 1031-54). We inactivated Rho in PC12 cells by scrape loading them with thebacterial toxin C3 before plating the cells on the test substrates. C3is known to inactivate Rho through ADP ribosylation (Udagawa, T. andMcIntyre, B. W. (1996) J Biol. Chem. 271, 12542-12548). On controlsubstrates of polylysine and laminin, treatment with C3 potentiated boththe number of cells with neurites and the length of neurites from cells(FIG. 3). On MAG and myelin substrates where neurite formation isinhibited, C3 has a dramatic effect on the ability to extend neurites(FIG. 3). When treated with C3 , about half of the PC12 cells plated oneither rMAG or native MAG has neurites of approximately 1 cell bodydiameter. In contrast, the untreated cells remained rounded and clumped.Similarly, PC12 cells plated on myelin remained rounded, but theaddition of C3 allowed neurites to extend directly on the myelinsubstrate. These results demonstrate that C3 treatment elicits neuritegrowth from PC12 cells plated on growth inhibitory myelin or MAGsubstrates.

Growth of Dominant-Negative Rho-Transfected Cells on MAG Substrates

PC12 cells transfected with constitutively active RhoA (V14GRhoA), andPC12 cells transfected with dominant negative RhoA (N19TRhoA), and themock-transfected cells, were examined for their ability to extendneurites on different test substrates. Cells with constitutively activemutation, V14GRhoA cells, differentiated poorly on all substrates,including poly-L-lysine and laminin. The treatment of the V14GRhoA cellswith C3 allowed the growth of some short neurites on all of the testsubstrates, including MAG.

In the same series of experiments the response of dominant negativeRho-transfected cells, N19TRhoA cells, to MAG and myelin substrates wasexamined. When N19TRhoA cells were plated on MAG substrates, they spreadand did not remain rounded as did the mock transfected PC12 cells. Asmall number of cells had short neurites, an effect that was observed onboth the rMAG and native MAG substrates (FIG. 3).

C3 treatment of mock transfected and N19TRhoA cells had a dramaticeffect on neurite outgrowth as most cells had extensive neurites (FIG.3). The effect of C3 on N19TRhoA cells was much more marked that theeffect on the mock transfected cells. Therefore, the combination of C3treatment and transfection of dominant negative Rho elicited excellentoutgrowth of neurites from PC12 cells plated on inhibitory MAG (andmyelin) substrates.

Effect of C3 on Primary Cells

To test the involvement of Rho in the response of primary neurons to MAGand to myelin substrates, cerebellar granule neurons were plated on testsubstrates and treated with C3 . Neurite outgrowth from these cells wasknown to be inhibited by MAG (Li, M., et al., (1996) J. Neurosic. Res.46, 404-414) and the C3 sitmulated growth of neurites from the granulecells on both permissive and inhibitory substrates.

The Growth Substrate Influences the Cellular Location of Rho

Rho is associated with the plasma membrane when it is in an activatedstate, and it moves into the cytosolic fraction when it is in theGDP-bound inactive state. To determine if the growth substrateinfluences the cellular localization of Rho, cells were either left insuspension or plated on MAG or collagen substrates, and preparedmembranes from the cells two hours later. It was shown that Rho wasprincipally localized in the cytosolic fraction when cells were platedon collagen, a growth permissive substrate. However, Rho was associatedwith the plasma membrane when cells where grown in suspension and whencells were plated on MAG (FIG. 4).

Example II In Vivo Demonstrations

1. Effect of C3 on Cultured Retinal Neurons

To test the involvement of Rho in the response of primary neurons to MAGand to myelin substrates, we purified retinal neurons and treated themwith C3 . Neurite outgrowth from these cells was inhibited by MAG (FIG.5 a). As with PC12 cells, treatment of retinal neurons cells with C3allowed neurite extension on the growth inhibitory MAG substrates to anextent similar to that observed on control substrates (FIG. 5 b and 5).

To ensure that the effect of C3 treatment resulted from uptake of C3into the cells, we examined by Western blot the electrophoretic mobilityof Rho in PC12 cells and retinal neurons treated with C3 (FIG. 6). Ithas previously been shown that ADP-ribosylation of Rho results indecreased mobility of Rho on SDS-acrylamide gels (Method Enzymol. Vol.256, Chapter 22 pg. 198). For our experiments, PC12 cells werescrape-loaded with C3 or with scrape-loading buffer as a control, andcells lysates were prepared after 48 hours in culture. Western blots ofthe lysates with anti-RhoA antibody revealed an increase in the apparentmolecular weight in cells treated with C3 . As a control for thespecificity of the effect, we probed the same blots for another smallGTPase of the Rho family, Cdc42. Cdc42 did not show any change inmobility upon treatment with C3 . To culture retinal neurons, retinaswere removed from P1-P5 rat pups, and the cells were dissociated with12.5 U papain/ml in Hanks balanced salts solution, 0.2 mg/ml DL cysteineand 20 μg/ml bovine serum albumin. The dissociated cells were plated ontest substrates in the presence of 50 mg/ml BDNF in DMEM with 10% FBS,vitamins, and penicillin/streptomycin in the presence or absence of 50mg/ml C3 transferase. Neurons were visualized by fluorescent microscopywith anti-β III tubulin antibody.

2. Effects of C3 on Retinal Ganglion Cell Axon Growth in Vivo

To explore the possibility that treatment of damaged axons with C3 mightfoster regeneration in vivo, we examined regeneration of retinalganglion cell (RGC) axons in the optic nerve 2 weeks after optic nervecrush. Recently, it has been shown that microlesions in the CNS reducethe extent of the glial scar and allow axons access to CNS white matterdistal to the lesion (Davies, S. J. A., et al. (1997) Nature 390,680-683). To make microlesions of optic nerve, 10.0 sutures were used toaxotomize RGC axons by constriction (FIG. 7 a). Retrograde labeling ofRGCs from the superior colliculus (not shown), as well as anterogradelabeling techniques (eg., FIG. 7 a) verified that RGC axons wereeffectively axotomized. To apply C3 to crushed nerves, Gelfoam soakedwith 2 mg/ml C3 was wrapped around the left optic nerve at the crushsite, and two Elvax tubes, each loaded with 20 mg of C3 , werepositioned for sustained slow release (FIG. 7 a). Twelve animals weretreated with C3 and a further 8 animals were treated with PBS ascontrols. Crushed and regenerating axons were visualized by anterogradelabeling with cholera toxin injected into the eye 12 days after opticnerve crush (FIG. 7 a). Fourteen days after optic nerve crush,longitudinal cryostat sections of the optic nerves were examined byfluorescent microscopy for immunoreactivity to cholera toxin to detectanterogradely labeled RGC axons.

In control optic nerves that received optic nerve crush alone, no RGCaxons extended past the crush site (n=3 animals). In control animalstreated with PBS-Elvax pellets and gelfoam, the crush site was easilydetected where most anterogradely labeled axons stopped abruptly (FIG. 7c). However, in these animals, a few axons did extend past the crush(FIG. 7 c, arrows), and the numbers of axons that regenerated variedfrom animal to animal. The application of Gelfoam and Elvax tubes mayhave altered the response to injury. Nonetheless, the response to C3treatment applied with this lesion paradigm was dramatic.

We observed that C3 treatment allowed many RGC axons to grow past theregion of the lesion. In 7 of 12 C3-treated animals, the lesion site wasnot clearly defmed because of the large numbers of axons that extendedthrough the site (FIG. 7 d and e). Many of the axons that extended pastthe lesion site showed a twist path of growth, supporting theiridentification as regenerating axons (FIG. 7 f) A quantitativecomparison of C3 and PBS treated animals revealed that more fibers grewpast the lesion site after C3 treatment than after PBS treatment (FIG. 7b). For this analysis we made a conservative estimate of the lesion sitebased on morphology, and counted the number of fibers in the distaloptic nerve in 14 μg sections. Seven of 12 C3-treated animals showed atleast one section with 20 axons extending 250 μm past the crush,compared with 1 of 8 of the PBS-treated controls (FIG. 7). In someanimals regenerating axons were observed up to 1 mm from the crush, anextent of regeneration similar to that observed in mouse optic nerveafter treatment with IN-1 antibody to block myelin inhibitors wherefibers extended upto 750 μm (Bartsch, U., et al., (1995) Neuron 15,1375-1381).

C3 Treatment of Crushed Optic Nerve in Adult Rats

Rats were anesthetized with 0.6 ml/kg hypnorm, 2.5 mg/kg diazepan and 35mg/kg ketamin. The left optic nerve was exposed by a supraorbitalapproach, the optic nerve sheath slit longitudinally, the optic nervelifted out and crushed 1 mm from the globe by constriction with a 10.0suture held for 60 seconds (FIG. 4 a). For C3 treatment and buffercontrols, Gelfoam soaked in PBS or 2 mg/ml C3 transferase was placed onthe nerve at the lesion site. Two 3 mm long tubes of Elvax (Sefton, etal., (1984) loaded with buffer or 20 mg C3 were inserted in the Gelfoamnear the nerve for continued slow release of C3 (FIG. 4 b). Twelve daysafter crush, 5 ml of 1% cholera toxinβ subunit (List Biologicallaboratories, Inc., Cambell, Calif.) was injected into the vitreous toanterogradely label retinal ganglion cell axons (FIG. 4 c). Two weeksafter optic nerve crush the animals were fixed by perfusion with 4%paraformaldehyde, and the eye with attached optic nerve was removed andpostfixed in 4% paraformaldehyde. Longitudinal cryostat sections wereprocessed for immunoreactivity to cholera toxin with goat anti-choleratoxin at 1:12,000 (List Biol. Labs Inc., Calif.) followed by rabbitanti-goat biotinylated antibody (1:200, Vector Labs, Burlingame,Calif.), and DTAF-streptavidin (1:500, Jackson ImmunoresearchLaboratories).

Discussion

Here we report that the small GTP binding protein Rho is likely to be akey intermediate in the neuronal response to neurite growth inhibitorysignals. Treatment of cultured PC12 cells, retinal neurons, andcerebellar granule cells with C3 enzyme to inactivate Rho allowedneurites to extend directly on inhibitor substrates of MAG or myelin.Also, PC12 cells transfected with dominant negative RhoA extendedneurites on MAG substrates. Therefore, inactivation of Rho wassufficient to allow neurite growth on MAG or myelin substrates whenneurons were grown in the presence of neurotrophic factors.

Further, our observations of microlesioned optic nerves after treatmentwith C3 provide the first evidence that the inactivation of Rho in axonsand non-neuronal cells near the site of lesion can help fosterregeneration after injury. While the in vitro experiments showed that C3can affect directly the growth of neurites from retinal cells, it islikely that the effects we observed after application of C3 to the opticnerve in vivo are more complex. C3 may affect other non-neuronal cells,such as macrophages and astrocytes, and these possibilities need to befurther examined. Nonetheless, our data provide compelling evidence thatC3 can promote neurite growth on inhibitory substrates in vitro, andhelps to overcome growth inhibition in vivo.

Regulation of Neurite Growth by Rho Family Members

Not all of the myelin-derived inhibitory molecules are known to date,and less is known about the neuronal receptors for growth inhibitorymolecules. Several different MAG receptors have been identified (Collinset al., 1997; Yang et al., 1996), and additional neuronal receptors tomyelin inhibitors are likely to exist. Targeting intracellular signalingmechanisms converging to Rho rather than individual receptors may be themost practical way to overcome growth inhibition in vivo. The advantageof inactivating Rho to stimulate regeneration is that axons canregenerate directly on the native terrain of the CNS, and thus may bemore likely to find their natural targets.

Both MAG and other myelin-derived growth inhibitory proteins block axonextension by causing growth cone collapse (Li, M., et al., (1996) J.Neurosci. Res. 46; 404-414; Bandtlow, C. E., et al., (1993) Science 259,80-83). These findings suggested to us that growth cone collapse by themyelin-derived inhibitors might be regulated by Rho. Moreover, innon-neuronal cells, Rho participates in integrin-dependent signaling(Laudanna, C., et al., (1996) Science 271, 981-983; Udagawa, T. andMcIntyre, B. W. (1996) J Biol. Chem. 271, 12542-12548). Together withthe observation that laminin can override myelin-derived inhibition, wehypothesized that small GTPases of the Rho family might play a role inintegrating singaling from positive and negative growth cues. Toinvestigate this possibility, we have made use of the ADP-ribosyltransferase C3 from Clostridium botulinum that efficiently inactivatesRho without affecting Rac and Cdc42, two other members of the Rho family(Udagawa, T. and McIntyre, B. W. (1996) J. Biol. Chem. 271, 12542-12548)and found that C3 treatment fosters neurite growth in the presence ofgrowth inhibitors. Moreover, immunocytochemical observations indicatethat Rho protein is concentrated at the filopodial tips of growth conesin adhesion structures called point contacts (Renaudin et al., 1998).Therefore, our in vitro results suggest the Rho signaling pathway is akey target for regulating growth cone motility and stimulatingregeneration.

Moreover, this data is relevant to the finding of Song et al. (Song etal., Science 281: 1515-1518 (1998)) who report that growth conerepulsion by MAG can be converted into attraction by elevation ofintracellular cAMP levels to activate protein kinase A (PKA).Experirnents with non-neuronal cells has implicated cAMP in theregulation of Rho because elevation of cAMP inhibits Rho activation(Laudanna, C., et al., (1996) Science 271 981-983). In PKA deficientPC12 cells, elevation of cAMP fails to protect from the activation ofRho by lysophosphatidic acid (Tigyi, G., et al., (1996) J. Neurochem.66, 537-548), a fmding that suggests that PKA-dependent regulation ofRho occurs in neural cells as well. Therefore, the cAMP-dependentregulation is likely to be upstream of Rho (Laudanna, C., et al., (1996)Science 271, 981-983).

The Non-Neuronal Response to Optic Nerve Injury

Remarkably, we observed that RGC axons crossed the lesion site to enterthe distal optic nerve after treatment of injured optic nerve with C3 .Some axons grew up to 1 mm past the site of lesion. This distance iscomparable to the maximal distances observed following treatment ofoptic nerve with IN-1 antibody (Bartsch, U., et al., (1995) Neuron 15,1375-1381). The most striking feature of our results was the largenumber of axons that were able to cross the lesion site compared toPBS-treated controls (see FIG. 7). Therefore, it is appears that C3 wasalso able to promote axon growth on inhibitory proteins present at theglial scar, indicating that targeting. the Rho signaling pathway aswidespread efficacy in stimulating axon regeneration after injury.

Example III

Inactivation of Rho or ROK Promotes Growth on Primary Neurons Plated onComplex Inhibitory Substrates

We tested if treatment of primary neurons with C3 or with Y27632(Y-27632) was sufficient to stimulate growth on complex inhibitorysubstrates typical of the glial scar and white matter. In thisparticular experiment, primary retinal neurons were isolated from P0-P3rats as described (Lehmann et al., . J Neurosci 19:7537-47, 1999). Testsubstrates were plated in 8-well chamber slides coated with 25 μg/mlpoly-L-lysine. Myelin substrates were made by coating with 8 μg purifiedbovine brain myelin dried overnight at room temperature. Chondroitinsulfate proteoglycan (CSPG) substrates were made by incubating 0.5 μg/mlmixed proteoglycans (Chemicon International, Inc. Temecula, Calif.)overnight in poly-L-lysine coated chamber slides. Dissociated cells werewashed, triturated with 25 or 50 μg/ml C3 or buffer, or with 35, 3.5 or0.35 μFM Y27632, and plated in culture medium with 50 ng/ml brainderived neurotrophic factor (BDNF) with or without C3 or Y27632. After 2days, the plates were fixed with 4% paraformaldehyde, 0.5%gluteraldehyde, and neurons were identified by immunocytochemistry usinga βIII tubulin antibody (Sigma, Oakville, Canada). Example ofpreparation of Y27632 has been disclosed in U.S. Pat. No. 4,997,834(Muro et al.,).

Neurons plated on chondroitin sulfate proteoglycans (CSPG) or purifiedmyelin had a rounded shape. After treatment with C3 or Y27632, neuronsplated on complex inhibitory substrates were able to extend neurites.Treatment either with C3 or Y27632 significantly increased the length ofneurites compared to untreated cells plated on myelin or CSPG. Theseresults demonstrate that inactivation of Rho or inhibition of ROKstimulates retinal neurons to extend neurites on growth inhibitorysubstrates. These results, illustrated in FIG. 8, were analyzedquantitatively by measuring the average of retinal ganglion cellsneurite length of the longest neurite per cell after 48 hours on PLL,CSPG or myelin including C3 or Y27632.

Treatment of Injured Spinal Cord Promotes Long Distance Regeneration

Balb-c female mice (n=70) of approximately 20 g were anaesthetized with0.4 ml/kg hypnorm and 5 mg/kg diazepam. A segment of the thoracic spinalcord was exposed using fine rongeurs to remove the bone, and a dorsalover-hemisection was made at T7. Fine scissors were used to cut thedorsal part of the spinal cord, which was cut a second time with a fineknife to ensure the lesion extended past the central canal. A fibrinadhesive delivery system was prepared using a Tisseel VH kit (ImmunoAG,Vienna, Austria). According to manufacturer's instructions for slowpolymerization, lyophilized fibrinogen was reconstituted in an aprotininsolution, thrombin was reconstituted in a calcium chloride solution, andboth solutions were warmed to 37° C. C3 (40 μg) or Y27632 (50 μg) wasadded to 25 μl of the thrombin solution. This was mixed with 25 μl ofthe fibrinogen solution just before application to the spinal cord toallow infiltration of the mixture into the lesion site beforepolymerization. In some animals, 10 μl of the 1 mg/ml C3 solution wasadded directly to the lesion site before injection of the C3 -containingfibrin adhesive. As controls, a second group of animals received fibrinadhesive alone after injury, and a third group was left untreated.Collagen gels with C3 were formed as follows. C3 was lyophilized (40 μgper mouse) then reconstituted in 10 ml of 7.5% NaHCO₃, and then 25 ml ofrat tail collagen at 0.7 mg/ml was added. Ten microliters of C3 wasadded to the lesion cavity before applying the C3 containing collagengel. For retransection of the spinal cord 3 weeks after SCI, the spinalcords were cut at T6 as described above, and the animals were observedfor changes in behavior by BBB testing for 1 week after the secondsurgery.

Anterograde labeling was performed as follows: three weeks to 3 monthsafter injury, the corticospinal tract (CST) fibers were labeled byinjection of the anterograde tracer WGA-HRP (wheat germagglutinin-horseradish peroxidase) into the motor cortex as described(Huang et al., Neuron 24:639-647, 1999). Two days later, the animalswere perfused transcardially with saline, then 4% paraformaldehyde, andthe spinal cords and brains were removed. Measurement of axonregeneration was determined from serial 30 μm cryostat sections assessedindependently by 2 reviewers.

To assess the potential of Rho inactivation to treat spinal cord injury(SCI), we cut the spinal cord of adult mice at T7 by a dorsalover-hemisection (Huang et al., Neuron 24:639-647, 1999). We testedlocal delivery of C3 in collagen (Joosten, J. Neurosci. Res. 41:481-490,1995) or in a fibrin adhesive (Herbert, 1998) that polymerizes in vivoseveral seconds after injection (Herbert, J. Biomed. Mater Res.40:551-559, 1998); Y27632 was tested in the fibrin adhesive. Anterogradetracing with WGA-HRP of corticospinal tract (CST), a tract often used tostudy histological regeneration, was used to assess fiber growth in sixgroups of animals: animals treated with fibrin plus C3 (n=13), collagenplus C3 (n=12), fibrin plus Y27632 (n=5), fibrin alone (n=10), collagenalone (n=7), and SCI with no treatment (n=13) (FIG. 9). Without C3 orY27632 treatment, transected CST axons retracted back from the site oflesion by approximately 300 μm, although in animals treated with fibrinalone some regenerative sprouts did extend from the retracted bundle.Application of C3 to the injured spinal cord elicited extensivesprouting into the dorsal white matter and the lesion scar. Treatedanimals with Y27632 showed regenerative sprouting into the dorsal whitematter and toward the lesion site. To assess axons distal to the lesionsite, the distance of the longest axon was measured. Axons were found upto 12 mm from the lesion site in C3 treated animals and up to 3 mm fromthe lesion site in Y27632 treated animals (FIG. 9), while buffer-treatedanimals showed retraction from the lesion site. Therefore, aftertreatment with C3 or with Y27632, axons were found to extend past thelesion into the distal white matter. These axons have a twisted courseof growth typical of regenerated axons.

Behavioral Testing

To test functional recovery after SCI and C3 or Y27632 treatment, wemeasured HL motor function using the Basso-Beattie-Bresnahan (BBB)locomotor rating scale (Basso et al., 1995) (n=37). Since a toeclearance phase cannot be evaluated in recuperating mice, we modifiedthe rating to a 17 point scale. Behavioral recovery was assessed for onemonth after SCI in an open field environment by the BBB method (Basso etal., 1995). We modified the 21 point BBB scale to a 17 point scorebecause mice do not exhibit differences in toe drag that can bemonitored visually. Thus, scale points 16, 17 and 18 were removed fromthe scale. Mice raise their tail early in their recovery, and score 19for tail up position was removed, leaving a 17 point total score. Themouse modified BBB score was as follows: [0] no observable hindlimb (HL)movement; [1] slight movement of one or two joints; [2] extensivemovement of one joint and/or slight movement of one other joint; [3]extensive movement of two joints; [4] slight movement of all threejoints of the HL; [5] slight movement of two joints and extensivemovement of the third; [6] extensive movement of two joints and slightmovement of the third [7] extensive movement of all three joints of theHL, walking with little/no weight support; [8] extensive movement of allthree joints, walking with weight support; [9] frequent to consistentdorsal stepping with weight support; [10] frequent plantar stepping withweight support; [11] consistent plantar stepping with weight support, nocoordination; [12] consistent plantar stepping with consistent weightsupport, occasional forelimb-hindlimb (FL-HL) coordination; [13]consistent plantar stepping with consistent weight support, frequentFL-HL coordination; [14] consistent plantar stepping with consistentweight support, consistent FL-HL coordination; predominant paw positionduring locomotion is rotated internally or externally, or consistentFL-HL coordination with occasional dorsal stepping; [15] consistentplantar stepping with consistent weight support, consistent FL-HLcoordination; predominant paw position is parallel to the body; frequentto consistent curled toes, trunk instability; [16] consistent plantarstepping with consistent weight support, consistent FL-HL coordination;predominant paw position is parallel to the body, flat toes, some trunkinstability; [17] consistent plantar stepping with consistent weightsupport, consistent FL-HL coordination; predominant paw position isparallel to the body, flat toes and consistent stability in thelocomotion. For scoring, each animal was videotaped for 3 minutes and 2reviewers participated. In the late phase of recovery, the BBB score wasdetermined from sequences of 4 steps or more from digitized videosprojected on a computer screen at 1/4 speed.

Twenty-four hours after surgery, control mice were paraplegic (FIG. 10)and moved by pulling themselves forward with their forelimbs. Micetreated with C3 or with Y27632 showed a remarkable recovery within 24hours (FIG. 10), already walking with weight support (FIG. 10). Whilethis early recovery is too rapid to be explained by long distanceregeneration, possible mechanisms include local reorganization ofcentral pattern generator circuitry (Ribotta et al., J Neurosci20:5144-52, 2000), pharmacological activation of neurotransmitterreceptors (Rossignol et al., Humana Press, Totowa. 57-87, 2000) orneuroprotection (Laufs et al., J Clin Invest 106:15-24, 2000; Trapp etal., Mol Cell Neurosci 17:883-94, 2001). Mice that had received C3 orY27632 treatment continued to recover over the 1 month period ofobservation, and exhibited hindlimb-forelimb coordination. By contrast,the average recovery plateau for untreated animals was limited tounstable walking without hindlimb-forelimb coordination. Retransectionof the spinal cord at 3 weeks (n=8) eliminated any achieved hindlimbrecovery in both C3 treated (n=5) and control (n=3) animals (data notshown).

1. An antagonist of one or more of Rho family members characterized bythe ability to elicit neurite outgrowth from cultured neurons in anassay method, comprising the steps of: (a) culturing neurons on a growthpermissive substrate that incorporates a growth-inhibiting amount of Rhofamily member; and (b) exposing the cultured neurons of step (a) to acandidate Rho family member antagonist agent in an amount and for aperiod sufficient prospectively to permit growth of the neurons; therebyidentifying as Rho family antagonists the candidates of step (b) whichelicit neurite outgrowth from the cultured neurons of step (a).